Protective Effect Of Bone Mesenchymal Stem Cell-Derived Microparticles Against H₂O₂-Induced H9C2 Cardiomyocyte Damage And Preliminary Study On Its Mechanism

Objective To investigate the effect of mesenchymal stem cell-derived microparticles against H2O2-induced cardiomyocyte injury,and to explore its preliminary mechanism.Methods The bone marrow mesenchymal stem cells was separated and purified by whole bone marrow adherence method.MSCs were observed by microscope,Flow Cytometry was used to analyze surface markers,cell counting chamber was used to draw growth curve,and MSCs was induced differentiation into adipocytes and osteoblast.These four methods are used to identify MSCs.The Fifth-generation MSCs were starved for 48 h,then the supernatant was collected,density gradient centrifugation was used to isolated microparticles derived from MSCs.BCA protein determination method was used to detect protein content,Flow Cytometry was used to analyze the size,amount,and surface markers of MSCs.After pretreatment with MSC-MPs(24 and 48 ?g·ml-1)for 24 h,H2O2(80 ?mol·L-1)acted on the cells for 3 h,and induced oxidative stress injury in H9C2 cells.Then,various indicators are tested,CCK-8 method was used to detect cell survival rate,the detection kit was used to detect the MDA,SOD,GSH and LDH content in the cells.The relative content of ROS and mitochondrial membrane potential were detected by using JC-1 detection kit and ROS detection kit,the Caspase-3 and Caspase-9 detection kits were used to detect the content of Caspase-3 and Caspase-9 in the cells,and real-time fluorescent quantitative PCR was used to detect the expression of PTEN?Bcl-2 and Bax m RNA in the cells.The experiment is divided into 5 groups:(1).Blank control group(Control): added cells culture medium and equal volume of PBS;(2).Cardiac cells injury model group(H2O2): added cell culture medium that contained H2O2(80 ?mol·L-1)for 3h;(3).MPs(24?g·ml-1)group: H9C2 cells were pretreated with MSC-MPs(24?g·ml-1)for 24 h,and then a cell culture medium containing H2O2(80 ?mol·L-1)was added.(4).MPs(48?g·ml-1)group: H9C2 cells were pretreated with MSC-MPs(48?g·ml-1)for 24 h,and then a cell culture medium containing H2O2(80 ?mol·L-1)was added.(5).Supernatant group(Supernatant): H9C2 cells were pretreated with an equal volume of supernatant for 24 h,and then a medium containing H2O2(80 ?mol·L-1)was added.Results 1.The experiment successfully isolated rat MSCs.The 5th generation of MSCs was spindle-shaped and typical "swarm-like" distribution under microscopy;the cells had strong proliferation ability;their surface markers were identified by flow cytometry,the positive rates of CD90 and CD29 were(99.81 ± 0.94)% and(99.90 ± 0.66)%,and the positive rates of CD45 and CD34 were(0.89 ± 0.31)% and(1.79 ± 0.33)%,respectively.Rat MSCs induced differentiate of osteogenesis and adipogenesis all were positive.2.MSC-MPs were separated by gradient centrifugation,and the number of MSCMPs was detected by flow cytometry.The results showed that the concentration of MSC-MPs was(2.55±0.86)×107cells/ml;the concentration of MSC-MPs protein was detected by BCA,and the concentration of MSC-MPs was tested before experiment.The surface markers of MSC-MPs were detected by flow cytometry,the experimental results showed that the positive rates of CD90 and CD29 were(97.46 ± 1.85)% and(94.00 ± 2.03)%,the positive rates of CD45 and CD34 were(4.89 ± 1.05)% and(1.81 ± 0.33)%,respectively,the MSC-MPs surface markers were similar to MSCs.3.H9C2 cells were treated with different concentrations of H2O2 for 3 h.The results of CCK-8 experiment showed that when the concentration of H2O2 increased,cell viability gradually decreased(P <0.05).When the cell survival rate was 46.12%(P <0.05),cell viability was close to 50%,so this concentration was selected as the proper concentration.4.The CCK-8 method was used to detect the effect of the cell survival rate after H9C2 oxidative stress injury.The experimental results show that MSC-MPs pretreatment(24?g·ml-1?48?g·ml-1)can significantly increase the cell survival rate from(46.96±4.94)% to(53.69 ± 3.06)% and(73.30 ± 4.84)%(P <0.05),but there was no significant change for the survival rate of H9C2 cells in the Supernatant group.5.The colorimetric method was used to detect the contents of LDH and SOD,MDA and GSH-Px in the supernatant or H9C2 cells.The MDA content in the cells was reduced from(1.98 ± 0.11)mmol·mg-1 to(0.97 ± 0.06)mmol·mg-1 and(0.86 ± 0.12)mmol·mg-1(P <0.05).The LDH content in supernatant decreased from(343.89 ± 11.43)U·L-1 to(288.39 ± 15.99)U·L-1 and(266.27 ± 12.93)U·L-1(P <0.05),and the SOD content in the cells increased from(184.78 ± 20.88)U·mg-1 to(259.27 ± 27.90)U·mg-1 and(334.88 ± 31.24)U·mg-1(P <0.05),the GSH-Px content in the cells increased from(139.09 ± 9.22)U·mg-1 to(170.48 ± 13.83)U·mg-1 and(177.21 ± 16.51)U·mg-1(P <0.05);for the supernatant group,LDH,MDA,SOD and GSH-Px had no significant changes compared with the H2O2 group.6.MSC-MPs(24?g·ml-1?48?g·ml-1)pretreatment can reduce ROS levels in H9C2 cardiomyocytes.The experimental results show that compared with the H2O2 group,the MSC-MPs pretreatment can reduce the intracellular fluorescence intensity,while the fluorescence intensity of the supernatant group had no significant change.7.MSC-MPs(24?g·ml-1 ? 48?g·ml-1)pretreatment can reduce the degree of mitochondrial damage.JC-1 fluorescence experiments showed that compared with the H2O2 group,MSC-MPs pretreatment can increase the ratio of Red/Green fluorescence intensity,but the ratio of Red/Green fluorescence intensity of the supernatant group had no significant change.8.The activity of Caspase-3 and Caspase-9 in H9C2 cells was detected by Caspase-3 and Caspase-9 detection kit.The experimental results showed that the levels of Caspase-3 and Caspase-9 in the H2O2 group were(151.76 ± 8.97)U·mg-1 and(205.83 ± 10.48)U·mg-1 respectively.MSC-MPs(48?g·ml-1)pretreatment can significantly reduce the levels of Caspase-3 and Caspase-9 to(118.28 ± 8.67)U·mg-1 and(174.42 ± 12.17)U·mg-1(P <0.05)respectively,but the levels of Caspase-3 and Caspase-9 in the supernatant group did not change significantly.9.The results of q RT-PCR experiments showed that compared with the H2O2 group,MSC-MPs(48?g·ml-1)pretreatment could reduce the expression of Bax and PTEN significantly,and increase the expression of Bcl-2.Conclusions 1.MSCs were successfully isolated from bone marrow and,MSC-MPs were successfully isolated from the supernatant of bone marrow mesenchymal stem cells;2.At 24 and 48?g·ml-1,MSC-MPs pretreatment improved cell survival rate,reducing the damage of H2O2 in H9C2 cells.It has been preliminarily confirmed that the protective effect is related to the inhibition of mitochondrial apoptotic pathway activation by inhibiting PTEN expression and reducing intracellular ROS levels.